comparison of several methods used for the determination of cephalosporins. analysis of cephalexin...

Journal of Pharmaceutical and Biomedical Analysis29 (2002) 405–423

Comparison of several methods used for the determinationof cephalosporins. Analysis of cephalexin in pharmaceutical

samples

Luisa Gallo Martınez, Pilar Campıns Falco *, Adela Sevillano CabezaDepartamento de Quımica Analıtica, Facultad de Quımica, Uni�ersidad de Valencia, C/Dr. Moliner 50, 46100 Burjasot,

Valencia, Spain

Received 19 December 2001; received in revised form 30 January 2002; accepted 17 February 2002

Abstract

The precision of UV absorbance of intac and acid degraded cephalosporins, ninhydrin, high performance liquidchromatography and iodometric methods used for analysis of cefoxitin, cefotaxime, cephazolin and cephalexin werecompared. To obtain the calibration graphs the analytical signal used were: absorbance, first derivative absorbance,second derivative absorbance and H-point Standard Additions Method by using absorbance values at two selectedwavelengths as analytical signal. These methods and calibration graphs were also used for the determination ofcephalexin in pharmaceutical samples. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cephalosporins; Ninhydrin; Spectrophotometry; High performance liquid chromatography; H-point Standard AdditionsMethod; Pharmaceutical samples

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1. Introduction

�-Lactam antibiotics have been used since thediscovery of penicillin in 1928. The penicillins andthe cephalosporins are both �-lactam.Cephalosporins have been used since 1948. Theseantibiotics have assumed a prominent role inmodern antimicrobial therapy due to enhancedintrinsic microbiological activities and favourablesafety profile. The 7-aminocephalosporanic acid(7-ACA) (Fig. 1) proceeds from the hydrolysis of

the cephalosporin C biologically active. Chemicalstructure of cephalosporins derive from the 7-ACA composed of a �-lactam ring fused with adihydrothiazine ring (Fig. 1), but differ in thenature of substituents attached at the 3- and/or7-positions of the cephem ring. These substitu-tions affect either the pharmacokinetic properties(3-position) or the antibacterial spectrum (7-posi-tion) of the cephalosporins [1]. Traditionally, thecephalosporins are divided into first-, second-,third- and fourth-generation agents [2,3]. Fig. 1shows the cephalosporins studied in this work.

Several methods have been reported forcephalosporin determinations. The official proce-dures in pharmaceutical preparations utilise iodo-

* Corresponding author. Tel.: +34-96-3983002; fax: +34-96-3864436.

E-mail address: [email protected] (P. Campıns Falco).

0731-7085/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0 731 -7085 (02 )00089 -4

mailto:[email protected]

L. Gallo Martınez et al. / J. Pharm. Biomed. Anal. 29 (2002) 405–423406

Fig. 1. Structure of the 7-ACA and structure general of the studied cephalosporins.

L. Gallo Martınez et al. / J. Pharm. Biomed. Anal. 29 (2002) 405–423 407

metric titration [4,5], the microbial assay [6], spec-trophotometric UV–vis analysis [7] and high-per-formance liquid chromatography (HPLC)methods [8]. The iodometric method is unselectiveas all antibiotics, the initial substances for theirproduction as well as their degradation products,are oxidised by iodine. It is therefore not suitablefor control of the purity of the antibiotics of thisgroup. The direct spectrophotometric methodssuffer a lack of specificity because all compoundscontaining the �-lactam ring absorb in the range250–270 nm. The major drawback of microbio-logical procedures is their lack of specificity instability studies when the decomposition productsare microbiologically active [9]. In order to im-prove the selectivity and sensitivity the HPLCwith ultraviolet absorption detection is themethod of choice frequently used forcephalosporin determinations in pharmaceuticalsamples. In most formulations the added ingredi-ents do not generally interfere with these methods.

Different reagents have been reported in orderto increase the selectivity of the methods based onthe own cephalosporins absorption or their prod-ucts of acid or basic degradation. Table 1 sum-marises the main parameters of different proposedmethods that use the spectroscopy UV–vis [10–32]. Hydroxylamine reacts with a number of car-boxylic acid derivatives to form hydroxamic acidsin presence of nickel (II) [10] or iron (III) [30] ascatalyst in the cephalosporins determination. Thepresence of amides, esters, aldehydes, ketones,anhydrides, etc. which also react with hydroxy-lamine, leads to positive errors in this determina-tion. The hydroxylamine method is more selectivethan iodometric or biological methods by less sothan HPLC methods. The ninhydrin reagent[11,17] is known to condense with thiophene andsome of its derivatives in the presence of concen-trated sulphuric acid to yield a coloured product[33]. Moreover, ninhydrin reacts with the 2-thienylacetic acid formed as a degradationproduct of cephalotin (analogous cephalosporinof cefoxitin) in a strong sulphuric acid medium[11]. In such a medium, cefoxitin also suffers ahydrolytic process [23]. Spectrophotometric deter-mination of cephaloridine, cefoxitin andcephalotin using ninhydrin in a strong sulphuric

acid medium is based on the presence of anunsubstituted thienyl moiety in both the �- and�-positions [17]. The determination ofcephalosporins by alkaline degradation to sul-phide hydrogen and formation of methylene bluehas been proposed [12]. Cephazolin [15] sufferedacid degradation when is heated in the presence ofsulphuric acid. This process can be observed bymeans of the disapparition of the band of 270 nmand the apparition of a new band at 302 nm dueto the hydrolysis product, 2-mercapto-5-methylth-iadiazole and there is also absorption at about250 nm by unidentified degradation products.Ammonium vanadate [14], cerium (IV) [32] andmolybdenum (VI) [16,20,22] have been used asoxidising agents in spectrophotometric determina-tions of �-lactam antibiotic. The excipients usu-ally added in capsules, such as starch, glucose andlactose interfere in this cephalosporins determina-tion [16]. 5,5�-Dithiobis(2-nitrobenzoic acid) (Ell-man’s reagent) [19] reacts with free thiol groupsbut a preliminary step of basic degradation isnecessary. Cephalosporins, being carboxylic com-pounds, react with 2-nitrophenylhydrazine hydro-chloride in presence of dicyclohexylcarbodi-imideand pyridine [24]. This method is general forcarboxylic compounds just as penicillins and itsdegradation products. The method of the imida-zole [25] is general for all cephalosporins contain-ing an intact �-lactam ring fused to asix-membered ring with sulphur atom. Hence canbe used as a stability indicating method and dif-ferentiates between the intact molecule and thedegraded one. The acetylacetone– formaldehydereagent method [26] is specific for amino �-lactamantibiotics. Non-amino �-lactam antibiotics andcommonly encountered excipients did not inter-fere. But this method would not be stability indi-cating, because it cannot detect the cephalexinprecursor, 7-amino-desacetoxy-cephalosporanicacid.

As can be seen in Table 1 the concentrationsare generally at mg/l levels. Most of them requirelong reaction times, between 15 and 90 min forthe ninhydrin and Ellman reagent, respectively,and temperatures from 30 to 100 °C.

Table 2 summarises some analytic properties ofthe HPLC methods [34–46]. These methods are


Tab

le1

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cpr

oper

ties

ofpr

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bien

tH

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Ni(

II)

as[1

0]am

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100

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104

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366

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Tab

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(con

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mol

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mol

cm


Tab

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(con

tinu

ed)

Rea

gent

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mpl

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(°C

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orsl

ope

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mol

cmph

orm

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dehy

de


Tab

le1

(con

tinu

ed)

Rea

gent

pHSa

mpl

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(°C

)�

orsl

ope

of�

(nm

)In

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rugs

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side

r-R

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ence

atio

nsde

tect

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calib

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ondy

nam

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me

(min

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oilin

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ater

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H0.

5–7

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lC

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les,

0.05

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n67

0.1

[27]

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lene

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bath

(hyd

roly

sis)

susp

ensi

on

Cep

hale

xin

Tab

lets

310

660

Nit

rate

of[2

8]co

balt

NaO

H

Cro

mot

rope

101.

860

0.4–

10�g

/ml

[29]

Cep

hale

xin

542

Cap

sule

s2B

102.

760

0.4–

14�g

/ml

564

Cro

mot

rope

2R

525

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mbi

ent

40�g

/ml

80–3

20�g

/ml

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hale

xin

510

�g/m

lH

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Fe

(III

)as

Pre

para

tion

[30]

cata

lyst

amin

eph

arm

aceu

tch

lori

de40

�g/m

l60

0�g

/ml

Cef

otax

ime

Cep

hale

xin

Pot

assi

umP

harm

aceu

t5–

100

�g/m

lF

irst

254

(Fir

st[3

1]de

riva

tive

)so

rbat

ede

riva

tive

prep

arat

ions

abso

rban

ce

15–4

5B

oilin

gw

ater

Cef

urox

ime

Am

mon

ium

0.9–

7.2

�g/m

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able

tsM

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2]ba

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ric

acid

sulp

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frequently used for cephalosporin determinationsin pharmaceutical samples in order to improvethe selectivity. The UV detectors are generallyused. The studied dynamic interval is in generalat mg/l levels.

In this paper, we compare different methodsproposed for the analysis of cefoxitin, cefo-taxime, cephazolin and cephalexin. Thesecephalosporins have also been assayed spec-trophotometrically after preliminary acid hydrol-ysis [15]. Acid degradation proceeds throughcleavage of the side chain amide linkage [47].The hydrolytic degradation of antibiotics is veryoften used as a preliminary step in the analyticalprocedures for their determination. The determi-nation of cephalexin in pharmaceutical prepara-tions is also studied.

On the other hand, the H-point Standard Ad-ditions Method (HPSAM) is a calibrationmethod that transforms the uncorrectable errorresulting from the presence of a direct interferentin the determination of an analyte into a con-stant systematic error. This error can then beevaluated and eliminated. This method also per-mits both proportional and constant errors pro-duced by the matrix of the sample to becorrected directly. The basis of the method forknown [48,49] or unknown [50,51] interferenceswere established.

We employed the calibration method HPSAMin order to test if the methods studied can beimproved in their selectivity/specificity.

2. Experimental

2.1. Reagents

Stock solutions of sodium cefoxitin (Merck,Darmstadt), sodium cefotaxime (Hoescht IbericaS.A.), sodium cephazolin (Lilly S.A., Alcoben-das, Madrid, Spain) and cephalexin hydrate(Sigma, St. Louis, MO) of pharmaceutical gradewere freshly prepared by dissolving 0.2000 g ofthe respective solid in 100 ml of distilled water(0.05 M sulphuric acid for cefotaxime). Stock-so-lutions of 7-ACA (Sigma) were prepared by dis-solving 0.2500 g of the solid in 100 ml of water.

The USP iodometric method provided the fol-lowing contents for three replicates 106�7% forcephazolin, 105�10% for cefoxitin and 107�10% for cefotaxime.

Working solutions were prepared by dilutionas required. Sulphuric acid 96% (Panreac,Barcelona, Spain), sodium hydroxide (Probus,Barcelona, Spain), sodium thiosulphate (Probus),potassium iodide (Scharlau, Barcelona, Spain)and iodine (D’Hemio, Madrid, Spain) were alsoused. All solutions were made in distilled waterand all reagents used were analytical-grade chem-icals.

For the application of HPLC method, stan-dard solutions of sodium cefoxitin, sodiumcephazolin, cephalexin hydrate and sodium ce-furoxime (Glaxo, Aranda of Duero, Spain) ofpharmaceutical grade were prepared by dissolv-ing 0.0158, 0.0158, 0.0161, 0.0105 g of the re-spective solid in 10 ml of distilled water. Thestock solution of sodium cefotaxime was pre-pared by dissolving 0.0263 g in 25 ml of distilledwater. Working solutions were prepared by dilu-tion as required.

Acetonitrile was HPLC grade from Scharlau.Water was distilled, deionized and filteredthrough 0.45-mm nylon membranes (Tec-knokroma, Barcelona, Spain). All the sampleswere filtered before their injection in the analyticcolumn with nylon filters (0.45 mm,Tecknokroma).

The NaH2PO4 solution was prepared by dis-solving 3.5 g of sodium dihydrogen phosphate(Probus) in 500 ml of distilled water. The pHwas adjusted to 3 by adding 50% H3PO4.

2.2. Mobile phase

A gradient of acetonitrile: NaH2PO4 (5×10−2

M, pH 3) with and increasing acetonitrile con-tent from 10% v/v at zero time, 20% v/v at 2min, 20% v/v at 6 min and 50% v/v at 8 min wasused. The solution was prepared daily, filteredthrough a 0.45-mm nylon membrane (Tec-knokroma) and degassed with helium before use.The flow rate was 0.75 ml/min. For quantitativedetermination a chromatographic signal wasmonitored at 254 nm. Nine replicates were made.


2.3. Dosage forms

They were obtained from local sources; severalformulations were used.

Kefloridina forte, 500 mg of cephalexin mono-hydrate for capsule (Lilly S.A.).

Kefloridina suspension, 250 mg of cephalexinmonohydrate for packet (Lilly S.A.).

Kefloridina mucolitico, 500 mg of cephalexinmonohydrate and 8 mg of bromhexine chlorhy-drate for capsule (Lilly S.A.).

Kefloridina mucolitico suspension, 250 mg ofcephalexin monohydrate and 4 mg of bromhexinechlorhydrate for packet (Lilly S.A.).

All solutions were made in water and allreagents were analytical-grade chemicals.

2.4. Equipment

Spectrophotometric measurements were made ina Perkin-Elmer Lambda 16 UV–vis double-beamequipped with 1-cm pathlength quartz cells andinterfaced to an Ataio S-2000 AT computer and anEpson LQ-400 printer. The spectra were recordedbetween 350 and 550 nm at 1 nm intervals.

A Hewlett-Packard HP 8452A diode-array spec-trophotometer equipped with a 1-cm pathlengthquartz cell and interfaced to a HP Vectra ES/12computer and HP Think-Jet printer was also used.The spectra were recorded between 350 and 550nm at 2 nm intervals.

pH measurements were made with a CrisonmicropH 2000 pH-meter (Crison Instruments,CORP., Alella, Barcelona, Spain).

A Hewlett-Packard 1040A liquid chro-matograph, equipped with a diode-array detector(Hewlett-Packard, 1040 Series), linked to a datasystem (Hewlett-Packard HPLC Chem Station,Palo Alto, CA) was used for data acquisition andstorage. The system was coupled to a quaternarypump (Hewlett-Packard, 1050 Series) and an auto-matic sample injector (Hewlett-Packard, 1050 Se-ries). The column was a Hypersil ODS-C18 5 m(125×4 mm2 i.d.) (Hewlet-Packard, Darmstadt,Germany). The detector was set to collect a spec-trum every 640 ms (over the range of 220–600 nm)and all the assays were carried out at room temper-ature.

To calculate all statistical least squared calibra-tion lines at all the measured wavelengths from thespectral data and the calculations of the HPSAM,a standard computer program package (StatView4.02 from ABACUS Inc.) for the Macintosh andthe Excel of Microsoft Office 97.

3. Procedures

These procedures were prepared according to[52,53].

3.1. Standard solutions of cepahlosporins

3.1.1. Assay of intact cephalosporinsDifferent volumes between 0.2 and 1.6 ml of

stock solutions of cephazolin and cefoxitin, andbetween 0.4 and 1.6 ml for the cefotaxime werediluted with distilled water (or acid sulphuric 0.05mol/l for the cefotaxime) to 100 ml. Differentvolumes between 0.1 and 1.6 ml of stock solutionsof cephalexin and between 0.2 and 1.6 ml of7-ACA were diluted with water to 50 and 10 ml,respectively. The absorbance between 210 and 400nm was recorded against distilled water.

3.1.2. Assay of degraded cephalosporinsThese products are prepare as in Ref. [23]: 20 mg

cephazolin, cefoxitin, cefotaxime and cephalexin,25 mg of 7-ACA or 10 ml of water for thepreparation of the blank, were transferred into 100ml volumetric flasks using 10 ml of 4.5 mol/lsulphuric acid. The solution was heated in a boil-ing water bath for 20 min, then it was cooled andneutralised with 6.0 mol/l sodium hydroxide solu-tion and made up the volume to the mark withwater (or sulphuric acid 0.05 mol/l for cefotaxime).Different volumes between 0.4 and 1.8 ml of stocksolutions of cephazolin, cefoxitin, cefotaxime werediluted appropriately to 50 ml. Different volumesbetween 0.2 and 1.6 ml of the stock solutions ofcephalexin and 7-ACA, respectively, or 5 ml of theblank solution were diluted to 25 ml. The ab-sorbance was registered as mentioned above.


3.1.3. Assay of cefoxitin with ninhydrin [54]The procedure used was that of Mahrous and

Abdel-Khalek [17]: different volumes of the stocksolutions of cefoxitin (4 mg of solid in 25 ml)between 0.10 and 1.00 ml and up to 1.00 ml ofdistilled water is added (if required), were trans-ferred into a 10 ml calibrated flask. A 0.10-mlvolume of 1% ninhydrin solution followed by 2.00ml of concentrated sulphuric acid were added.The solution was shaken gently and was leftstanding for 5 min at room temperature. Thecontents were diluted to 10 ml with 50% sulphuricacid. After 5 min, the absorbance between 300and 600 nm was recorded against distilled water.

3.1.4. Assay of degraded cefoxitin with ninhydrinThe procedure used is that was described in

Ref. [23]: 200 mg of cefoxitin was transferred intoa 100 ml calibrated flask using 10 ml of 9 Msulphuric acid. The solution was heated in a boil-ing water bath for 20 min, then cooled and neu-tralised with a 9 M sodium hydroxide solution.The volume was filled up to the mark with water.10 ml of this stock solution was diluted to 50 ml.Different volumes of this solution were trans-ferred into a 10 ml calibrated flask, and thesubsequent experimental procedure followed theabove guidelines for calibration graphs of cefox-itin. The absorbance was registered as mentionedabove.

3.2. Applications to pharmaceutical samples [53]

3.2.1. Preparation of sample

3.2.1.1. Intramuscular injections. The stock solu-tions of each cephalosporin were prepared dis-solving the appropriate quantities of the powderin 100 ml of distilled water.

3.2.1.2. Capsules. Thoroughly mix the contents of5 capsules, weigh, transfer an accurately weighedquantity of powder equivalent to 183 mg ofcephalexin to a 100 ml, volumetric flask, anddissolve in and dilute to volume with water. Shakewell and filter through Whatman No. 42 paper.Discard the first portion of filtrate. Use the clearsolutions obtained as stock solutions (0.005 mol/l).

3.2.1.3. Oral suspension. Accurately weigh a quan-tity of powder equivalent to 183-mg cephalexin,and treat as described under capsules.

3.2.2. Degradation of samples

3.2.2.1. Intramuscular injections. As it is indicatedin Section 3.1.2 [44].

3.2.2.2. Capsules and oral suspension. Accuratelyweigh a quantity of powder equivalent to 286-mgcephalexin, and prepare a solution as describedabove. Add to 10 ml of this solution 10 ml of 9.0mol/l sulphuric acid, and treat the solution asdescribed for the degraded cephalosporins [23].

3.3. Method of the ninhydrin

The products of reaction of the cefoxitin withninhydrin were prepared as indicated in Section3.1.3.

The products of the reaction of the cefoxitindegraded with ninhydrin were prepared as indi-cated in Section 3.1.4.

3.4. High-performance liquid chromatography

The pharmaceuticals samples of the cephalexinwere prepared as indicated above. The solutionsof Kefloridina forte capsules, Kefloridina oral sus-pension, Kefloridina forte mucolitico capsules andKefloridina suspension oral mucolitico were pre-pared dissolving 0.0030, 0.0333, 0.0034 and 0.0333g, respectively, in 25 ml of distilled water.

4. Results and discussion

The precision of different methods used for thedetermination of cefoxitin, cephazolin, cefotaximeand cephalexin alone and in the presence ofbromhexine is evaluated. Fig. 2 shows the per-centage recovery�standard deviation calculatedfor cefoxitin when different methods are em-ployed. The wavelengths utilised for intact cefox-itin are: 240 nm for absorbance; 244 and 280 nmfor first derivative absorbance; 236, 252, 270 and292 nm for second derivative absorbance; and for


HPSAM the pairs of � are 240–276, 224–280 and250–272 nm. For acid degraded cefoxitin thewavelengths utilised are 240 nm for absorbance,248 nm for first derivative absorbance, and 240,256 and 270 nm for second derivative absorbance.Using ninhydrin method, the wavelengths are 454nm for absorbance, and 424 and 474 nm for firstderivative absorbance. According to fundamentalsof the HPSAM applied to analytical proceduresthat use a reagent blank, two wavelengths areselected in which the external and internal blanksshow the same absorbance [55,56]. The wave-lengths selected are 456–476 nm and 444–492nm. The results of HPLC method are obtained at254 nm.

Good precision is obtained working with theform intact. The worst results are obtained work-ing with the degraded form and using the secondderivative.

In a previous paper [54], we shown that differ-ent products were obtained from cefoxitin withand without hydrolysis by sulphuric acid andreaction with ninhydrin. An in situ calibration isread in order to prevent the possible formation ofacid degraded cefoxitin–ninhydrin product when

it is working with the intac antibiotic. Because ofthis when using the ninhydrin method the bestresults are obtained applying the HPSAMmethod. It may be that the influence of the aciddegraded cefoxitin–ninhydrin compound hasbeen eliminated. This influence is more importantwhen the measured cefoxitin concentration issmaller. The precision obtained by the HPSAM/ninhydrin is similar to those obtained usingHPLC method and iodometric method.

Table 3 gives the relative recovery for all meth-ods tested for intact and degraded cephazolin.The worst precision corresponds to iodometricmethod and second derivative using Lambda 16spectrophotometer. Good results are obtainedwith the others methods.

As can be seen in Table 4 for intact and de-graded cefotaxime, the worst precision is obtainedusing iodometric method. The others results ob-tained are suitable. From Table 5 for cephalexin,similar conclusion can be derived.

The precision obtained when these methods areapplied to the determination of cephalexin in dif-ferent pharmaceutical samples is similar to thatobtained for cephalexin standard. In a previous

Fig. 2. Recovery percentage�standard deviation found in the analysis of cefoxitin using different methods; number of replicates isthree for each result. Range concentrations: (1.04–8.19)×10−5 mol/l for intact, and acid degraded, (0.36–3.56)×10−5 mol/l usingninhydrin method and (0.06–117)×10−5 mol/l for HPLC. 4.45×10−3 mol/l for iodometric method. The various symbols (�–�)show different concentrations included in the range studied.

L. Gallo Martınez et al. / J. Pharm. Biomed. Anal. 29 (2002) 405–423416T

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Fig. 3. Average recovery percentage found for three replicates of each result using different methods of determination of cephalexinin capsule. The range of concentrations studied were: (1.06–27.95)×10−5 mol/l and (2.03–36.56)×10−5 mol/l for cephalexin alone(a) and in presence of bromhexine (b) respectively. The various symbols (�–�) show different concentrations included in the rangestudied.

paper [52], we found that the excipients usuallyadded in pharmaceutical samples, such as starch,talc, dicalcium phosphate and lactose did notdisturb these determinations. Fig. 3 and Fig. 4show the average recovery percent obtained whenusing different methods for the analysis of phar-maceutical samples of cephalexin. When HPSAMis applied to the determination of intactcephalexin the pairs of � are 224–280, 208–230and 212–280 nm. Whereas, for the degraded are230–288 and 224–276 nm.

The wavelengths utilised when the others meth-ods are applied are shown in Table 5 for intactand acid degraded standards.

As can be seen in Fig. 3 and Fig. 4 good resultsare obtained using all the methods for the deter-mination of intact and degraded cephalexin aloneand in presence of bromhexine. The best accuracyis obtained using HPSAM.

5. Conclusions

This paper shows several methods for the deter-mination of intact and degraded cephalosporins.It is demonstrated that the intact cephalosporinsdetermination provides, generally, better results

than those estimated by acid degraded compounddetermination. But the direct spectrophotometricmethods suffer a lack of specificity because allcompounds containing the �-lactam ring absorbin the range 250–270 nm. Besides, the latter meth-ods can improve selectivity because the impurity7-ACA that can be present in the trade productalso shows less interference. Also, acid degradedcephalosporins spectra present more significancedifference than intact drugs spectra.

When using the ninhydrin method, the applica-tion of the HPSAM reduces the blank bias errorand the interference of other species. HPSAMpermits the determination of cefoxitin in presenceof degraded cefoxitin. 7-ACA does not bear the2-thienyl moiety and failed to condense with nin-hydrin under the conditions of determination ofcefoxitin. HPSAM also provides a precision simi-lar to HPLC and iodometric methods for thiscephalosporin.

For cephazolin, cefotaxime and cephalexin theprecision achieved by intact or acid degradedUV–vis spectrophotometry and HPLC was goodand similar. Worse precision was obtained byiodometric method.

Good results are obtained in the analysis ofintact cephalexin in all pharmaceutical samples.


Fig. 4. Average recovery percentage found for three replicates of each result using different methods of determination of cephalexinin suspension. The range of concentrations studied were: (1.06–28.83)×10−5 mol/l and (2.163–30.49)×10−5 mol/l for cephalexinalone (a) and in presence of bromhexine (b) respectively. The various symbols (�–�) show different concentrations included in therange studied.

However, working with degraded cephalexin thecalibration method should be optimised. HPSAMis the best options for pharmaceuticals tested.

Acknowledgements

The authors are grateful to the DGICYT forthe financial support (Project no. PB 97-1387).

References

[1] L. Balant, P. Dayer, R. Auckenthaler, Clin. Pharma-cokinet. 10 (1985) 101.

[2] J.D. Williams, Drugs 34 (1987) 15.[3] N.C. Klein, B.A. Cunha, Med. Clin. N. Am. 79 (1995)

705.[4] B. Pospisilova, J. Kubes, Cesk. Farm. 38 (1989) 136.[5] B. Borowiecka, G. Pajchel, W. Chojnowsky, Acta Pol.

Farm. 46 (1989) 463.[6] U.S. Pharmacopeia, 22nd Rev., U.S. Pharmacopeial Con-

vention, Inc., Rockville, MD, 1990.[7] U.S. Pharmacopeia, 22nd Rev., U.S. Pharmacopeial Con-

vention, Inc., Rockville, MD, 1985.[8] U.S. Pharmacopeia, 22nd Rev., U.S. Pharmacopeial Con-

vention, Inc., Rockville, New York, 1990, pp. 251–252.[9] R. Wise, P.J. Wills, J.M. Andrews, K.A. Bedford, An-

timicrob. Agents Chemother. 17 (1980) 84.[10] D.L. Mays, F.K. Banger, W.C. Cantrell, W.G. Evans,

Anal. Chem. 47 (1975) 2229.

[11] P. Papazova, P.R. Bontcher, M. Kacarova, Pharamazie32 (1977) 486.

[12] M.A. Abdalla, A.G. Fogg, C. Burgess, Analyst 107 (1982)213.

[13] M.A. Abdalla, A.G. Fogg, C. Burgess, Analyst 108 (1983)53.

[14] M.M. Abdel-Khalek, M.S. Mahrous, Talanta 30 (1983)51.

[15] P. Papazova, P.R. Bontcher, M. Kacarova, Talanta 30(1983) 51.

[16] M.M. Abdel-Khalek, M.S. Mahrous, Talanta 31 (1984)635.

[17] M.S. Mahrous, M.M. Abdel-Khalek, Analyst 109 (1984)611.

[18] F.A. El Yazbi, M.H. Barary, Anal. Lett. 18 (1985) 629.[19] F.I. Sengun, I. Fedai, Talanta 33 (1986) 366.[20] P.B. Issopoulos, J. Pharm. Biomed. Anal. 6 (1988) 97.[21] B. Morelli, Analyst 113 (1988) 1077.[22] P.B. Issopoulos, J. Pharm. Biomed. Anal. 7 (1989) 619.[23] M.A. Korany, M. Abdel-Hady Elsayed, S.M. Galal,

Anal. Lett. 22 (1989) 159.[24] M.A. Korany, M. Abdel-Hady Elsayed, M.M. Bedair,

A.A. Gazy, Talanta 36 (1989) 1253.[25] A.A. Alwarthan, S. Abdel Fattah, N.M. Zahran, Talanta

39 (1992) 703.[26] I.T. Patel, B. Muljibhai, B. Devani, T.M. Patel, JAOAC

Intern. 75 (1992) 994.[27] A.A. Alwarthan, F.H. Mctwally, S.A. Al-Tamimi, Anal.

Lett. 26 (1993) 2619.[28] A.A. Alwarthan, H.A. Ai-Lohedan, Talanta 41 (1994)

225.[29] Y.M. Issa, A.S. Amin, Mikrochimica Acta 124 (1996)

203.


[30] D. Agbaba, S. Eric, K. Karljikovic-Rajic, S. Vladimirov,D. Zivanov-Stakic, Spectros. Lett. 30 (1997) 309.

[31] S. Trajkovic-Jolevska, A. Dimitrovska, Anal. Lab. 7(1998) 148.

[32] A.F.M. El-Walily, A.A.K. Gazy, S.F Belal, E.F. Kamis,Spectros. Lett. 33 (2000) 931.

[33] F. Feigl, Spots Test in Organic Analysis, seventh ed.,Elsevier, Amsterdam, 1966.

[34] R.P. Buhs, T.E. Maxim, N. Allen, T.A. Jacob, F.J. Wolf,J. Chromatogr. 99 (1974) 609.

[35] M.E. Rogers, M.W. Adlard, G. Saunders, G. Holt, J.Chromatogr. 297 (1984) 385.

[36] J.H. Kennedy, B.A. Olsen, J. Chromatogr. 389 (1987)369.

[37] C.F. Martin, L. Takahashi, J. Worsley, C. Hagemeier, L.Hall, J. Chromatogr. 402 (1987) 376.

[38] C.M. Moore, K. Sato, Y. Katsumata, J. Chromatogr. 539(1991) 215.

[39] B.A. Olsen, S.W. Baertschi, R.M. Riggin, J. Chromatogr.648 (1993) 165.

[40] C. Garcıa, J.L. Perez, B. Moreno, Analyst 120 (1995) 53.[41] M. Hsu, Y. Lin, H. Chung, J. Chromatogr. 692 (1995) 67.[42] P.S. Mukherjee, H.T. Karnes, Analyst 121 (1996) 1573.[43] Z.J. Wu, W.B. Guo, Q.G. Zhang, K.Y. Ni, Y.S. Lin,

Sepu 17 (1999) 518.

[44] J.L. Ming, H.Q. Zhu, Yaowu-Fenxi-Zazhi 20 (4) (2000)271.

[45] J.L. Ming, H.Q. Zhu, Yaowu-Fenxi-Zazhi 20 (2) (2000)130.

[46] S.G. Kaskhedikar, A. Argal, Ind. Drugs 38 (3) (2001)137.

[47] C.O. Wilson, O. Gisvold, R.F. Doerge, Organic Medici-nal and Pharmaceutical Chemistry, eighth ed., 1982, p.251.

[48] F. Bosch, P. Campins, Analyst 113 (1988) 1011.[49] F. Bosch, P. Campins, Analyst 115 (1990) 111.[50] F. Bosch, P. Campins, J. Verdu, Anal. Chim. Acta 283

(1993) 831.[51] P. Campins, J. Verdu, F. Bosch, C. Molins, Anal. Chim.

Acta 302 (1995) 323.[52] P. Campıns, L. Gallo, A. Sevillano, F. Bosch, Anal. Lett.

29 (11) (1996) 2039.[53] P. Campıns, A. Sevillano, L. Gallo, F. Bosch, I. Monzo,

Mikrochim. Acta 126 (1997) 207.[54] P. Campıns, A. Sevillano, L. Gallo, F. Bosch, Anal.

Chim. Acta 334 (1996) 199.[55] P. Campins, F. Bosch, J. Verdu, Anal. Chim. Acta 270

(1992) 253.[56] F. Bosch, J. Verdu, P. Campins, C. Molins, Talanta 41

(1994) 39.

comparison of several methods used for the determination of cephalosporins. analysis of cephalexin...

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